Method for determining the presence or concentration of a bound enzyme

ABSTRACT

There is disclosed a process and a device for detecting and measuring (1) the amount of enzyme present as a detecting system following a nucleic acid hybridization reaction or immunoreaction; (2) the level and activity of free enzyme in a biological sample; (3) the level of enzyme from contaminating microorganisms present in a sample; and (4) enzymes from pure culture isolates for microbial identification and antimicrobial susceptibility testing.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method and a device for the rapid andsensitive detection and measurement of an enzyme which (1) is used aspart of the detection or reporting system for an immunoassay or nucleicacid hybridization assay, (2) is present in a biological sample, (3) isassociated with a microorganism for detection of the microorganism in asample, and (4) is used for pure culture tests, such as microbialidentification and antimicrobial susceptibility tests.

BACKGROUND OF THE INVENTION

Nucleic acid hybridization and antibody immunoassay technologies havebeen developed that permit rapid, sensitive and specific measurements oforganic compounds and microorganisms. Recent advances have been directedtoward improving the sensitivity and specificity of these assay systemsby enhancing the detection or "reporting" of the antigen-antibodycomplex or nucleic acid hybrid duplex which is formed. Many approacheshave been attempted in this regard. One such example is the multiplelabeling of an antigen, antibody or nucleic acid probe with an enzyme toproduce a nonisotopic and highly sensitive diagnostic test. For example,multiple copies of enzyme can be chemically coupled to a molecule ofavidin. The avidin can then bind strongly to biotin, which has beenchemically inked to an antibody, antigen or a nucleic acid probe. Theresult is the presence of multiple copies of enzyme for everyantigen-antibody complex or nucleic acid hybrid formed. Another approachused in nucleic acid hybridization assays is the use of multipleenzyme-labeled probes that hybridize to different sequences on thetarget genome. Whichever approach is used to amplify the biologicalsignal, the result of the assay is usually determined by the developmentof a distinct color or fluorescence that is read visually or with aninstrument.

The detection of specific nucleic acid sequences through the use ofhybridization probes is a well established procedure. One commonly usedmethod involves the immobilization of the target polynucleotide sequenceon a solid support (e.g., nitrocellulose, diazobenzyloxymethylcellulose, nylon, etc.). The immobilized nucleic acid is then denatured,if it is double stranded, and subsequently hybridized to a complementaryprobe. The probe nucleic acid sequence is labeled isotopically, usuallywith ³² p, or nonisotopically with direct labeling of the polynucleotidesequence with an enzyme or indirectly with a biotin-avidin system.

In contrast to radioisotopically labeled probes, nonisotopic systemsoffer advantages of safety, relatively low cost, and ease of use-However, enzyme detection often suffers from high background values fromthe nonspecific adsorption of labeled probes to the solid support.Non-specific adsorption may be reduced with multiple washing steps,which add to the length and difficulty of the procedure.

A different method for detecting a specific polynucleotide sequenceinvolves the displacement of a labeled nucleic acid, according to themethod of Vary ,et al., "Nonisotopic Detection Methods for StrandDisplacement Assays of Nucleic Acids," Clin. Chem. 32:1696-1701 (1986).A labeled polynucleotide "signal strand" is hybridized with a largersequence (the "probe strand"), which is, in turn, complementary to thetarget polynucleotide sequence of interest. Interaction of the targetsequence with the signal-probe hybrid results in the displacement of thesignal strand from the hybrid. After separating the displaced signalstrands from the signal-probe hybrid, the signal strand is measuredusing an isotopic label such as ³² p or nonisotopic labels such as anenzyme. Such assays are potentially more sensitive because of thereduction of background signal due to nonspecific adsorption.

Two types of enzyme immunoassays are commonly used. The sandwichimmunoassay involves the capturing of antigen molecules in a solution bysolid phase-bound antibody molecules. A second antibody molecule, whichis enzyme-labeled and specific to a different antigenic determinant, issubsequently added to the solid phase-bound antigen-antibody complex.Similarly, the competition immunoassay involves the competition ofantigens for antibody binding sites. Enzyme-labeled antigen andunlabeled antigen from the sample (the antigen of interest) compete forbinding sites on the solid phase bound antibody. In these cases, theamount of enzyme remaining on the solid support is either proportional,in the first example, or inversely proportional in the second example,to the amount of antigen in the sample.

Attempts at increasing the sensitivity of enzyme immunoassays (EIA) andhybridization assays have frequently focused on increasing the amount ofproduct generated per antigen-antibody complex or hybrid formed byincreasing the number of labeled enzyme molecules. Enzyme amplificationoften results in an increase in false positive reactions due toincreased nonspecific adsorption or an increase in false negativereactions due to inhibition of antigen and antibody binding orhybridization by complementary polynucleotide sequences.

Little effort has been directed towards increasing assay sensitivity byenhancing the measurement of the signal or "product" that is generatedby the enzyme reacting with the substrate. Frequently, the assaysensitivity is reduced because of a high background-signal. Themeasurement of extremely low levels of colored or fluorescentenzyme-generated product by an instrument is often compromised by theinherent color or fluorescence of the substrate. This problem can befurther exacerbated by the common use of high concentrations ofsubstrate to accommodate a low binding affinity of the enzyme.Background signal can also result from assay and sample components thatare colored, fluorescent, luminescent or electrochemically active. Inmost cases, a positive result is reported only when the enzyme-generatedsignal is twice the background signal.

In addition to the use of enzymes for detecting immunoreactions andhybridization reactions, little progress has been made for increasingassay sensitivity for detecting free enzymes in a sample as well asenzymes produced by microorganisms. Assays to measure and detect freeenzymes and microbial enzymes in a biological sample generally utilizesubstrates that produce enzyme-generated products that are colored,fluorescent, luminescent or electrochemically active. The sensitivity ofthese assays is most hindered by a high background signal from sampleconstituents and assay components including substrate.

One attempt to enhance the measurement of an enzyme-generated productwas described by Kiuchi et al. (A Fluorometric Microassay Procedure forMonitoring the Enzymatic Activity of GM1-Ganglioside-B-Galactosidase byUse of High-Performance Liquid Chromatography, 1984, Anal. Biochem.140:146-151). These investigators utilized a high performance liquidchromatography (HPLC) system to measure theGMi-ganglioside-β-galactosidase activity in crude tissue samples bymeasuring increased NADH concentration. The biological steps of thisprocedure, including the incubation of sample with substrate, wereconducted in a vessel separate and apart from the HPLC instrument.Following incubation of the substrate and enzyme from the sample, thereaction solution was injected into an HPLC instrument which separatedthe various assay components. The disadvantage of this procedure is thata conventional HPLC column with a high number of theoretical plates isrequired to sufficiently separate the components. This means that theseparation procedure of Kiuchi et al. is a lengthy procedure andrequires the use of an expensive HPLC instrument which is capable ofmoving fluids through the large column at high pressures, often inexcess of 3,000 psi. The column used by Kiuchi et al. had the dimensionsof 4 mm×300 mm and was packed with reverse phase C18 particles. A columnof this type will typically have in excess of 15,000 theoretical platesat optimal linear efficiency.

Wehmeyer et al. (Liquid Chromatography with Electrochemical Detection ofPhenol and NADH for Enzyme Immunoassay, 1983, J. Liquid Chromatography6:2141-56) refers to an enzyme immunoassay procedure with a smaller HPLCcolumn with the dimensions of 50 mm×2 mm to separate phenol from othercomponents in the reaction solution. Phenol was generated by theenzymatic cleavage of phenylphosphate. Similar to the procedure atKiuchi et al., Wehmeyer et al. performed the enzyme immunoreaction in avessel separate from the HPLC instrument. After sufficient incubationtime for the enzyme and substrate in this vessel, the reaction solutionwas injected into the HPLC instrument. Wehmeyer et al. needed a longHPLC column to accomplish sufficient separation of phenol. The problemwith a long HPLC column is an increase in analysis time and the requireduse of HPLC rated components which can handle high pressure as a resultof the use of a long column. Also, extraneous materials in the reactionsolution can potentially co-elute with phenol resulting in a significantreduction in, overall assay sensitivity and specificity.

Therefore, there is a need in the art for a method and device toincrease the sensitivity of non-isotopic immunoassays and nucleic acidhybridization assays that use enzymes for reporting assay results.Additionally, there is a need in the art for methods for measuring anddetecting free enzymes from microorganisms in a sample and frommicroorganisms in pure culture.

DISCLOSURE OF THE INVENTION

Briefly stated, the present invention discloses methods and associateddevices for enhancing the detection of a bound marker enzyme which hasbeen generated by an immunoreaction or by a hybridization reaction in anassay system. The method generally comprises: (a) adding a substrate ina selected solution to a first column containing a complementary (to thesubstrate) marker enzyme bound to a solid phase; (b) incubating thefirst column to enzymatically convert the substrate to a product in anamount proportional to the amount of enzyme present; (c) transferringthe product and unreacted substrate onto a second column of no more than500 theoretical plates and preferably approximately 100 theoreticalplates, and most preferably about 50 theoretical plates, the secondcolumn containing a sorbent capable of selectively binding the productin the presence of the selected solution; (d) selectively eluting theproduct from the solution; and (e) detecting the presence orconcentration of the product. The product may be detected by measurementof absorbance, fluoresence, luminescence or by electrochemical activityand may be adapted to a continuous flow or stop flow cycleconfiguration. The sorbent may selectively retain the product throughpolar or non-polar interactions, ion exchange, other specific molecularinteractions such as affinity binding, or combinations thereof. Themethod may be performed using a manual format or with an instrument witha fluidics system with a continuous flow or stopped flow configuration.

The result is a greatly reduced or completely eliminated backgroundsignal. Also, the enzyme-generated product is concentrated from a largereaction volume to a small detection volume. Further, the use of asorbent bed with minimal column length and theoretical plates results ina low pressure system, a rapid separation of product from other assaycomponents, and a reduction of reagent requirements.

In a related aspect of the present invention, the device generallycomprises: (a) a first column containing an enzyme bound to a solidsupport, the enzyme present from an immunoreaction or a nucleic acidhybridization reaction; and (b) a second column connected in series tothe first column and containing a sorbent bed of no more than 500theoretical plates and preferably approximately 100 theoretical platesand most preferably approximately 50 theoretical plates, wherein thesorbent is capable of selectively binding an enzymatically generatedproduct which has been produced through the addition of a substratespecific to the enzyme in the first column. In a related aspect of thedevice, a detection device is connected in series to the second column,the detection device being capable of measuring the amount of producteluted from the second column. The use of connected columns eliminatesthe necessity of performing an enzyme immunoassay or hybridization assayin a separate vessel and then injecting the reaction solution into asecond detection instrument.

In a further related aspect of the present invention, the devicecomprises a second column containing a sorbent bed of no more than 500theoretical plates, and preferably approximately 100 theoretical platesand most preferably, 50 theoretical plates, wherein the sorbent iscapable of selectively binding an enzymatically generated product, saidproduct is produced through the addition of a substrate specific to thefree enzyme of interest in a sample, or to enzymes produced bymicroorganisms of interest in a sample, or in a pure culture. In arelated aspect, a second column is connected in series to a detectiondevice capable of measuring the amount of product eluted from the secondcolumn containing the sorbent.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1c depict various arrangements of the two columns. Column 1(the first column) is used to retain the solid support materials uponwhich the nucleic acid hybridization reaction or immunoreaction isperformed. Column 2 (the second column) is used to contain the sorbentmaterial. The two columns may be separated with a switching valvelocated between the columns as indicated in FIG. 1a or the columns maybe present in a single unit but separated by a barrier, such as a fritor membrane, as indicated in FIG. 1b. A multiple column arrangement maybe configured into an injection molded plastic unit with flow connectorsbetween columns (FIG. 1c). The paths by which fluids flow through thecolumns and connectors is determined by switching valves locatedexternal from the plastic unit.

FIG. 2 details a system design for a microprocessor-controlledinstrument that utilizes pressurized gas to move reagents and sample.Metering pumps can be substituted for the pressurized gas. FIG. 2illustrates the use of a plastic unit as detailed in FIG. 1c withswitching valves located external to the plastic unit to direct flowthrough the columns.

FIG. 3 illustrates a manual plastic disposable device with a two-columnarrangement for enzyme detection and measurement. In this case, the twocolumns are separated by a frit. Two directional movement of fluidsthrough the columns is accomplished by manual movement of the plasticpart containing the columns in and out of the receiving vessel. A rubber"O" ring located on the insertion end of the unit with the columns formsa seal with receiving vessel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a method and a device for increasing thesensitivity of immunoassays or nucleic acid hybridization assays. Thedevice includes two columns connected in series, permitting low pressuremovement of solutions through columns; the first column is used toperform a solid phase immunoreaction or nucleic acid hybridizationreaction and the second column is packed with sorbent particles. Thefirst column contains the means for conducting an immunoreaction ornucleic acid hybridization reaction or the means for capturing solidsupport materials such as latex beads upon which the immunoreaction orhybridization reaction has been performed. Similarly, the product of theimmunoreaction (antigen-antibody complex) or hybridization reaction(probe-target duplex) which is formed in solution outside of the firstcolumn, may be retained on the first column by physical means such as onthe surface of a membrane or by chemical means such as throughcovalently binding onto an affinity membrane (e.g., ULTRABIND™, GelmanSciences, Ann Arbor, Mich.), through nonspecific binding ofantigen-antibody complexes by Protein A coated to a solid supportmaterial, or through an antigen (or hapten)-antibody reaction on a solidsupport material. Once the enzyme is present on the first column as aresult of the immunoreaction or hybridization reaction, a substratespecific to the enzyme is added to the first column under time andincubation conditions sufficient to allow the enzyme and substrate toreact to form a product. The product of the enzyme reaction is producedin proportion to the amount of enzyme present as a result of theimmunoreaction or hybridization reaction.

Turning to the drawings, FIG. 1 depicts various arrangements of the twocolumns. In FIG. 1A, the first column 2 retains the solid supportmaterial upon which the nucleic acid hybridization reaction orimmunoreaction will be performed. The second column 4 contains thesorbent material. The two columns may be separated with a switchingvalve 6 located between the columns as indicated in FIG. 1A, or thecolumns may be present in a single unit but separated by a barrier suchas a frit 8 or membrane as indicated in FIG. 1B. A multiple columnarrangement may be configured into an injection molded plastic unit 10with flow connectors 12 between columns, as depicted in FIG. 1C. Themolded plastic unit 10 has a width 14, which is preferably about 5inches, and a height 16, which is preferably about 2 inches. The pathsby which the fluid flows through the columns and connectors isdetermined by switching valves located externally to molded plastic unit10.

FIG. 2 details a system designed for a microprocessor-control instrumentthat utilizes pressurized gas to move reagents and sample, coupled withthe use of a molded plastic unit 10 as detailed in FIG. 1C withswitching valves located external to the plastic unit to direct flowthrough the columns. Metering pumps can be substituted for thepressurized gas. The flow of sample and reagents is controlled by apressure regulator 24, which controls pressure through a two-way gasmanifold 22. Pressure is transmitted to pressure vessels 26 for reagentdelivery. The pressure may be released through manifold 23. The reagentsare conducted through and mixed in a second manifold 20, and sample 30is then mixed with the reagents. The resulting mixture is then conductedthrough a third manifold 28 and pumped to molded plastic unit 10. Aftera sufficient period of time, the desired constituents are then conductedthrough a fourth manifold 31 to a detector 32.

FIG. 3 illustrates a manual plastic disposable device with a two-columnarrangement for enzyme detection and measurement. The manual unitcomprises a threaded sample container 38, a threaded cap 36 and areceiving vessel 40. In this embodiment, the first column 2 and thesecond column 4 are separated by a frit 8. Two-directional movement offluid through the columns is accomplished by manual movement of theplastic part containing the columns in and out of the receiving vessel40. A rubber "O" ring 42 located on the insertion end of the unit havingthe columns form a seal with the receiving vessel 40.

Briefly, the first column contains an enzyme bound to a solid support asthe result of a nucleic acid hybridization reaction or immunoreaction. Asubstrate, which is specific for the enzyme and is in a solution whichpermits substantial or complete retention of the enzyme-generatedproduct onto the sorbent in the second column, is added to the firstcolumn and allowed.-to incubate under appropriate conditions. Thesolution containing the product and unreacted substrate is then allowedto flow into the second column using an instrument-based fluidicssystem, such as the one shown in FIG. 2, or using a manual format asshown in FIG. 3. The second column functions to bind the product thatflows through it, through use of the sorbent. Preferably, the substratepasses substantially through the column. The ability of the sorbent tobind the product is dependent upon the chemical nature of the solutioncarrying the product and substrate. This allows the product to beconcentrated on the sorbent contained within the second column.Subsequent elution results in the concentration of the product,substantially free of substrate and other extraneous materials, into avolume generally smaller than the original volume of solution applied tothe second column. The use of a second column with a low number oftheoretical plates, as a function of column length and inner diameter,permits the use of a low pressure diagnostic test that can be performedrapidly and in a manual format, or with a low pressure instrument. Thedetector for the instrument may be located downstream from the secondcolumn, as is shown in FIG. 2.

In another embodiment, the assay system is used for (i) the detection offree enzymes of interest in a sample, (ii) for detection of microbialcontaminants of interest in a sample, and (iii) for identification andantimicrobial susceptibility testing of pure culture microorganisms. Forthe detection of free enzymes, the assay is performed by adding a sampleto an assay solution containing a substrate, and incubating at anappropriate temperature. The free enzymes specifically chemically modifythe substrate to produce a product that can be retained on the sorbentbed.

An example of a free enzyme determination in a biological sample is thedetection of alkaline phosphatase in fluid milk. A loss of alkalinephosphatase activity in milk is indicative of the effectiveness ofpasteurization of fluid milk. A fluorogenic substrate for alkalinephosphatase, 4-methyl-umbelliferyl phosphate (MUP) is added as asubstrate to a sample of fluid milk. If the alkaline phosphatase enzymeis present, it cleaves the MUP to produce the productmethylumbelliferone (MU). MU can be concentrated on a sorbent bed asdescribed herein, and then measured by fluorescence detection.

Similar to the free enzyme assay, the detection of microbialcontaminants in a sample involves incubation of the sample at anappropriate temperature in an assay solution containing a substrate. Theassay measures the amount of enzyme produced by the microorganisms inthe sample to detect and estimate the level of microorganisms. Theseenzymes may be present within the cell or released from the cell. Forexample, coliform bacteria produce the enzyme β-galactosidase and E.coli produce the enzyme glucuronidase. The incubation of a substratesuch as 4-methylumbelliferyl-β-D-galactoside (MUGAL) with a samplecontaining coliform bacteria or the incubation of the substrate4-methylumbelliferyl-β-D-glucuronide (MUG) with a sample containing E.coli can produce the fluorescent product MU in proportion to the levelsof these microorganisms in the sample.

Identification and antimicrobial susceptibility tests can be performedon a suspension of a pure culture isolate. Substrates and other reagentsfor these pure culture tests may be similar to those used in EuropeanPatent No. EP-A-91,837 and by Snyder and Wang ("Rapid Characterizationof Microorganisms by Induces Substrate Fluorescence: A Review,"Biotechnology Progress 1:226-230, 1985) and by Snyder, et al. ("PatternRecognition Analysis on In Vivo Enzyme Substrate Fluorescence Velocitiesin Microorganism Detection and Identification," Appl. Environ.Microbiol. 51:969-977). Useful substrates include indoxyl acetate,indoxyl-β-D-glucoside, 4MU-D-Glucoside, 4MU-Phosphate, Indoxylphosphate, 4-MU-D galactoside, N-methyl indoxyl acetate, N-methylindoxyl myristate, β-naphthyl acetate, α-naphthyl acetate,4MU-heptanoate, 4MU-acetate, 5-cromoiodoxyl acetate,5-bromo-4-chloro-3-phosphate, 3-indoxyl phosphate,6-bromo-2-naphthyl-β-D-glucoside, 4MU glucuronide, 7-ethoxycoumarin,glycyl-L-phenyl-β-naphtyylamide, β-naphthyl sulfate, 3-indoxyl sulfate,luminol, resazurin, fluorogenic 4-methylumbelliferyl derivatives,derivatives of 7-amino-4-methylcoumarin, a mixture of4-methylumbelliferyl phosphate and 4-methylumbelliferyl fatty acid estersuch as the hexonate, octanoate, nonanoate or other fatty acid esterwith a chain length of C₆ -C₁₆, and a mixture of 4-methylumbelliferylester such as phosphate and7-(N)-(aminoacyl-4-peptidyl)-4-methyl-7-amino-coumarin (such as7-(N)-alanyl-4-methyl-7-amino coumarin and the corresponding leucinederivative. Useful fluorogenic substrates include peptides and esters(in themselves known materials) of umbelliferone, 4-methylumbelliferone,3-carboxy-7-hydroxycoumarin, 3-acetyl-7-hydroxy coumarin,3-carboxyethyl-7-hydroxycoumarin, 3-cyano-7-hydroxycoumarin,7-amino-4-methylcoumarin, 7-amino-4-trifluoromethylcoumarin,2-naphthylamine, 4-methoxy-β-naphthylamine, naphthol-AS(3-hydroxy-2-naphthoic acid anilide), indoxyl 1-(alpha)-and2-(beta)naphthol derivatives including N-methyl indoxyl acetate andindoxyl acetate, resorufin, 1-methyl-7-hydroxyquinolinium iodide, and6-amino-quinoline. For microbial identification, the culture suspensionis added to solutions, each containing a different substrate, andincubated at an appropriate temperature. Multiple enzyme tests may beformatted into a panel of tests. Depending upon the type ofmicroorganism, certain substrates will be cleaved by specific enzymesproduced by the microorganism to produce a characteristic pattern. Theproduct of the enzyme reaction may be fluorescent, colored,electrochemically active or luminescent. Fluorogenic substrates such as7-amino-4-methylcoumarin are derivatized to amino acids such as alanine,leucine and valine or to short-chain peptides or proteins or4-methylumbelliferone derivatized to compounds such as acetate,propionate and phosphate, fatty acids such as oleate and heptanoate, andsugars such as galactose, fucose, arabinose and glucose. When thesefluorogenic substrates are hydrolyzed by the microbial enzymes,4-methylumbelliferone (MU) and 7-amino-4-methylcoumarin (AMC) areliberated.

The products are fluorescent and will strongly bind to a C18 sorbent.The AMC and MU can be eluted from the C18 sorbent with 100% methanol andcan be measured with a fluorometer.

Recognition of the specific enzymes that a particular microorganismproduces permits identification of the pure culture isolate.Antimicrobial susceptibility patterns can be obtained by measuring areduction in the amount of enzyme produced by a pure culture isolate inthe presence of certain antimicrobial compounds. Reduction in the amountof enzyme produced by the pure culture isolate is directly related tothe susceptibility of that microorganism to the antimicrobial.Fluorogenic substrates such as 4-methyl-umbelliferyl phosphate (MUP),4-methylumbelliferyl nonanoate (MUN) andL-alanyl-7-amido-4-methylcoumarin (AAMC) can be used for theantimicrobial susceptibility testing. A reduction in the amount offluorescent product produced, MU and AMC, from enzymatic cleavage, isindicative of a decrease in the level of enzyme related to microbialinhibition by the antimicrobial.

In each of these cases, the incubated solution is added to a secondcolumn containing the sorbent. The second column contains a sorbent of,at most, 500 theoretical plates and preferably approximately 100theoretical plates and most preferably approximately 50 theoreticalplates. The second column functions to bind the product formed byincubation of the substrate with the biological sample. Separation ofthe product from the substrate is achieved as described herein.

As noted above, the immunoreaction or hybridization reaction can beperformed on a solid support, such as latex beads or sephacryl beads orlatex-coated frits within the first column. Also, the immunoreaction orhybridization reaction can be performed outside of the first column on asolid support system, with the first column being used to retain thesolid support after completion of the immunoreaction or hybridizationreaction. Further, the products of the immunoreaction (antigen-antibodycomplex) or hybridization reaction (hybrid duplex) can be retained onthe first column by physical or chemical means. A detection system isprovided through use of the enzyme label which is directly andchemically linked to an antigen, antibody or nucleic acid probe, orindirectly labeled to any part of the assay reactants using, forexample, avidin-biotin binding. The enzyme is part of theantigen-antibody complex or the hybrid duplex if the immunoreaction orhybridization reaction is completed outside of the first column.Immunoreactions or hybridization reactions conducted on a solid supportor in solution outside of the first column can involve multiple manualsteps, including the sequential addition of reagents, incubations andwashings. These steps may be performed automatically by a programmedinstrument. For example, the addition of reagents to the solid phasesupport system, the washing and the incubation steps may all beconducted by a microprocessor-controlled fluidics system.

In the case of an immunoreaction that is performed on the surface of asolid support, antigen or antibody is covalently attached or passivelyadsorbed to the surface of the solid support. If an antibody is attachedto the support, then the antigen of interest in the reaction sample canbe detected with a competition immunoreaction or a sandwichimmunoreaction- In the case of a competition immunoreaction, anenzyme-labeled antigen (the antigen is identical to the antigen ofinterest) is mixed into the sample to be tested. This mixture is thenpassed over the antibody-coated surface where the unlabeled antigen andthe enzyme-labeled antigen compete for binding sites on the surface ofthe solid phase. The amount of enzyme remaining on the solid supportsurface provides a quantitative measurement of the level of unlabeledanalyte or antigan of interest in the sample.

In the case of a competition immunoreaction performed in solution,unlabeled antigen and a standardized amount of enzyme-labeled antigenare mixed with a standardized concentration of antibody in an assaysolution. The concentration of the enzyme-labeled antigen and antibodyshould be standardized such that the ratio of antigen determinant toantibody binding site is approximately 1:1. After the immunoreaction iscompleted, the assay solution is introduced into Column 1 containingsolid support material coated with Protein A. The Protein A binds to theFc portion of the antibody molecule and retains the immune complex thathas been formed in solution. The amount of enzyme remaining attached tothe Protein A solid support is inversely proportional to the amount ofunlabeled antigen of interest in the sample.

In a sandwich-type immunoreaction, the antibody molecules on the solidsupport surface bind to the specific antigan of interest in the sampleto be tested. Unreacted antigan and extraneous materials are thenremoved through a wash step. A second antibody molecule, often specificfor a different antigenic determinant on the antigen molecule, is enzymelabeled and added to the solid phase system. The solidphase-antibody-antigen complex will bind the antibody-enzyme. Throughthis mechanism, the enzyme label is bound to the solid support as partof an antibody-antigen-antibody sandwich-type complex. The amount ofenzyme remaining attached to the solid phase after washing isproportional to the amount of antigen in the sample of interest. Thesolid support can be part of the first column. Alternatively, the solidsupport can be a latex bead which is later collected in the first columnafter completion of the immunoreaction.

In the case of a hybridization reaction, oligonucleotide strands thatare specific for and complementary to the target DNA or RNA arecovalently connected to the surface of the solid phase. Preferably, theoligonucleotide strands are connected to the solid phase at one end ofthe oligonucleotide strand. If a sandwich-type hybridization reaction isperformed, DNA or RNA from the microorganism or another source to betested is chemically or mechanically released, denatured, and hybridizedto the strands on the solid phase under appropriate solution conditions.An enzyme labeled nucleic acid probe that reacts specifically to adifferent base sequence of the captured target sequences is thenpermitted to hybridize to the complementary nucleic acid strand on thetarget strand. This enzyme labeled probe is captured by the targetnucleic acid sequence on the solid phase and acts to report the presenceof the target strand on the solid phase. Therefore, with a hybridizationreaction, the presence of an enzyme bound to the solid phase serves toindicate the presence or absence of specific nucleic acid sequences ofinterest.

A competition nucleic acid hybridization reaction is performed by usingan enzyme labeled nucleic acid probe that is complementary to the strandof nucleic acid on the solid phase. The enzyme-labeled probe alsocontains a nucleic acid sequence that is identical to or substantiallysimilar to a target sequence of interest on a polynucleotide strand froma biological sample. The enzyme labeled nucleic acid probe is mixed withthe target nucleic acid from the sample of interest. Both the labeledand the unlabeled target sequence compete to hybridize to complementarysequences bound to the solid phase. The amount of enzyme remaining onthe solid phase is inversely proportional to the amount of targetnucleic acid sequence in the sample of interest.

A hybridization reaction can also be performed in solution and thehybrid captured in the first column. An example is the capturing of anucleic acid hybrid formed in solution with an antigen and antibodyreaction in the first column. In this case, two polynucleotide probeswhich are complementary and specific to two unique sequences on a targetpolynucleotide strand are used. One of these probes is labeled withenzyme and the second probe is labeled with hapten. The probes hybridizewith the target strand under appropriate assay conditions. After thehybridization reaction is completed, the assay solution containing thehybridization complex is introduced into the first column which containssolid support material coated with antibodies specific to the hapten.The antibodies retain the hybridization complex. This results in thepresence of enzyme on the first column.

A second example involves the use of antibodies that specifically reactwith a DNA:DNA hybrid or a RNA:DNA hybrid. In this case, one of thecomplementary strands is labeled with enzyme. As was described above,after hybridization in solution is completed, the assay solutioncontaining the hybrid is introduced into the first column containingsolid support materials coated with antibodies to the DNA:DNA hybrid orRNA:DNA hybrid. This retention of the DNA:DNA or RNA:DNA hybrids resultsin the presence of enzyme in the first column, the amount of enzymepresent in the first column is proportional to the amount of targetnucleic acid in the sample of interest.

As briefly described above, the first column may contain the solid phasematerial upon which the immunoreaction or hybridization reaction isperformed to generate a bound marker enzyme. Alternatively, the firstcolumn may be used to retain the solid phase materials from animmunoreaction or hybridization reaction which had been performedoutside of the first column to capture the marker enzyme. The retentionof the solid phase materials in the first column can be accomplishedwith a physical barrier such as a frit or membrane at one opening of thefirst column. Further, the first column may act to capture anantibody-antigen complex, or a probe-target oligonucleotide duplex, bothcontaining an enzyme, formed in solution outside of the first column.Each alternative described above results in the presence of a reportingenzyme bound to a solid phase material in either direct or inverseproportion to the molecule, antigen, nucleic acid or microorganism ofinterest. The present invention discloses a process and a device foramplifying the signal generated by the enzyme by reducing the backgroundsignal and by concentrating the product into a smaller detection volume.

As noted above, once the enzyme label is bound to the solid support, aspecific substrate, in a solution which permits complete binding of theenzyme-generated product, is added to the first column. The substratereacts with the bound enzyme, thereby generating a product. Theappropriate reaction conditions, such as time, temperature and pH areadjusted in accordance with the characteristics of the specific enzymeinvolved. Preferably, the substrate is present in a solution whichallows (a) optimal enzyme activity; (b) complete binding of theenzyme-generated product on the sorbent; and (c) little or no binding ofthe substrate on the sorbent. Many enzymes show high activity inbuffered solutions such as 0.01-0.1M phosphate buffer (pH 6-8),0.001-0.1M Tris buffer (pH 6-8), 0.001-0.1M borate buffer (pH 6-9) or0.001-0.1M bicarbonate buffer (pH 7-9). A further description of theretention of the enzyme-generated product relative to sorbent andreaction solution is provided below.

After incubation, the solution containing the unreacted substrate andthe product generated from the reaction of the substrate with the boundmarker enzyme is transferred to a second column. The fluidic design forthe solution transfer between columns permits direct contact of the twocolumns separated by a permeable device, or by a conduit between columnsor by a microprocessor-controlled fluidics device that can movesolutions from a first column to a second column.

The second column functions to substantially separate the product fromthe substrate in solution. The second column contains a sorbent thataffects this separation by retaining the enzyme-generated productrelative to the unreacted substrate and other contaminants. Thisseparation enhances the detection of extremely low amounts of enzymebound to the solid support in the first column.

The sorbent may be any material that can separate the product fromsubstrate. Suitable sorbents generally affect this separation throughpolar, non-polar or ion interactions with the product. The completeretention of the enzyme generated product on a particular sorbent withlittle or no retention of substrate is dependent upon the type ofsorbent, the chemical nature of the product and substrate, and thesolution used to conduct the enzyme reaction. Table 1 illustrates theinterdependence of these components.

                                      TABLE 1                                     __________________________________________________________________________                    Chemical                                                                      Nature of                                                                            Chemical                                                     Functional                                                                              Enzyme-                                                                              Nature of                                                                            Preferred                                       Classes of                                                                          Groups on Generated                                                                            Specific                                                                             reaction                                        Sorbents:                                                                           Sorbent   Product:                                                                             Substrate:                                                                           Solution:                                       __________________________________________________________________________    Non-polar                                                                           Octadecyl (C18).sup.1                                                                   Substantially                                                                        Substantially                                                                        Polar                                                 Octyl (C8)                                                                              non-polar                                                                            polar  solution                                              Ethyl (C2)              (generally                                            Cyclohexyl (CH)         under 0.1M)                                           Phenyl (PH)                                                             Polar Cyanopropyl (CN)                                                                        Substantially                                                                        Substantially                                                                        Non-polar                                             Diol (20H)                                                                              polar  non-polar                                                                            solution                                              Silica (SI)                                                                   Aminopropyl (NH.sub.2)                                                        N-propylethylene                                                              diamine (PSA)                                                           Ion-  Benzene-  Negatively                                                                           Substantially                                                                        pH between                                      exchange                                                                            sulfonyl- or positively                                                                        uncharged or                                                                         pKa's of                                              propyl (SCX)                                                                            charged                                                                              has a counter                                                                        product                                               Sulfonylpropyl   charge to                                                                            and sorbent;                                          (PRS)            the product                                                                          low ionic                                             Carboxymethyl           strength                                              (CBA)                                                                         Diethylamino-                                                                 propyl (DEA)                                                                  Trimethyl-                                                                    aminopropyl                                                                   (SAX)                                                                   __________________________________________________________________________     .sup.1 Types of functional groups that are chemically linked to the           particulate support such as silica are indicated as well as common            commercial designations for sorbents with these functional groups.       

The description of a non-polar sorbent for retention of anenzyme-generated product is provided for illustration. Preferably, anon-polar sorbent is utilized, such as a bonded C8 to C22 silica. Mostpreferably, the non-polar sorbent is a C18 silica sorbent, a C18 styrenedivinyl/benzene, or a C18 alumina. Non-polar sorbent separates productfrom substrate based upon differences in the relative degree ofhydrophobicity. The C8 to C18 non-polar sorbents are commonlyconstructed using activated silica or alumina with carbon chains ofvarious lengths extending from the surface. The numeric designation ofC8 or C18, for example, refers to the number of carbon atoms in thechain. The carbon chain creates a non-polar region around the bondedsilica. Compounds that are substantially non-polar in nature, orcompounds that contain non-polar regions are added to the sorbent bed ina solution that is as polar as possible, such as water. In the situationof a polar solvent, the substantially non-polar compounds will associatewith the non-polar regions of the sorbent. When non-polar sorbents areused, the enzyme-generated product and substrate should be moved to thesorbent bed in the second column by a solvent that is essentially polar.

A compound will elute off of the sorbent bed when the compound is moreattracted to the eluting solvent than to the sorbent. The sorbent andthe solvent for a particular product and substrate are chosen such thatthe product is retained on the sorbent bed until the elution solvent isadded to the second column. An increased assay sensitivity is realizedfrom the elution of the product from the sorbent bed into a very smallvolume of solvent.

The chemical nature of the substrate and product dictate the appropriatetype of sorbent and solvent or reaction solution used. For example,Table 2 below lists examples of enzymes and substrates and resultingproducts generated. Table 2 also lists appropriate combinations ofsorbent and eluting solvent that can be used to separate the productfrom the substrate.

                                      TABLE 2                                     __________________________________________________________________________                      Retained     Eluting                                        Enzyme   Substrate                                                                              Product                                                                              Sorbent                                                                             Solvent Detector                               __________________________________________________________________________    β-Galactosidase                                                                   4-Methylum-                                                                            Methylum-                                                                            C-18  Methanol                                                                              Fluoro.                                         belliferyl-                                                                            belliferone                                                          β-D-Galactoside                                                 β-Glucuronidase                                                                   4-Methylum-                                                                            Methylum-                                                                            C-18  Methanol                                                                              Fluoro.                                         belliferyl-                                                                            belliferone                                                          β-D-Glucuronide                                                 Glucosidase                                                                            4-Methylum-                                                                            Methylum-                                                                            C-18  Methanol                                                                              Fluoro.                                         belliferyl-                                                                            belliferone                                                          α-D-Glucoside                                                  Alkaline 4-Methylum-                                                                            Methylum-                                                                            C-18  Methanol                                                                              Fluoro.                                Phosphate                                                                              belliferyl                                                                             belliferone                                                          phosphate                                                            Protease 4-Methylum-                                                                            Methylum-                                                                            C-18  Methanol                                                                              Fluoro.                                         belliferyl                                                                             belliferone                                                          casein                                                               Esterase 4-Methylum-                                                                            Methylum-                                                                            C-18  Methanol                                                                              Fluoro.                                         belliferyl                                                                             belliferone                                                          laurate                                                              Glucose- NAD+     NADH   C-18  Methanol                                                                              Fluoro                                 6-Phosphate                            meter or                               Dehydrogenase                          electro-                                                                      chemical detector                      β-Galactosidase                                                                   Nitrophenyl-                                                                           Ortho- C-18  Acetonitrile                                                                          spectro-                                        thio-β-D-                                                                         Nitrophenol          photometer or                                   Galacto-                      electro-                                        pyranoside                    chemical detector                      Alkaline p-Nitro- p-Nitro-                                                                             C-18  Acetonitrile                                                                          spectro-                               Phosphatase                                                                            phenyl   phenol               photometer or                                   phosphate                     electro-                                                                      chemical detector                      Lactate  Lactic Acid                                                                            Pyruvic                                                                              Cation                                                                              Salt    Fluoro.                                Dehydrogenase                                                                          NADH     Acid   Exchange                                                               NAD+                                                        Peptidase                                                                              Peptide  Free Amino                                                                           C-18/ Methanol/                                                                             Fluoro.                                         Chain    Acids w/                                                                             Anion Salt                                                             addition of                                                                          exchange                                                               OPA (ortho-                                                                   ophthaldial-                                                                  dehyde)                                                     Aminopeptidase                                                                         L-Arginine                                                                             Amino- C-18  Methanol                                                                              Fluoro.                                B        Aminomethyl                                                                            methyl-                                                              coumarin coumarin                                                    Pyruvate ADP and  ATP'   Ion   acidic  Lumino-                                kinase   phosphoenol                                                                            pyruvate                                                                             exchange                                                                            ammonium                                                                              meter                                           pyruvate        (NH.sub.2)                                                                          phosphate                                                                     buffer                                         __________________________________________________________________________

Pyruvate Kinase's enzyme generated product, ATP, is mixed with theenzyme luciterase, or the ATP is passed through a column which containsimmobilized luficerinase, to generate light, which is measured by a flowluminometer.

For a particular type of sorbent, the appropriate selection of thesubstrate-enzyme combination and the reaction solution (solvent) isessential for the practice of this invention. The substrate and enzymegenerate a product which is retained strongly on the sorbent and ismeasurable with a detection device (fluorometer, spectrophotometer,luminometer, electrochemical detector). Further, the enzyme generates aproduct which is substantially chemically distinct from the substrate.For example, the substrate may contain both polar and non-polar groups.The enzymatic cleavage of this compound produces a measurable productwhich is substantially non-polar in nature. An example of thissubstrate-enzyme combination is the substrate 4-methylumbelliferylB-D-galactoside which has both a non-polar moiety (themethylumbelliferyl group) and a polar one (D-galactose). This compoundis enzymatically cleaved by the enzyme B-galactosidase to producegalactose and methylumbelliferone. The methylumbelliferone moiety isnon-polar and fluorescent. Similarly, the substrate might besubstantially ionic (positively or negatively charged), whereas theproduct produced by enzyme activity has a countercharge to the substrateor has no charge. This product can be separated from the substrate by anion exchange sorbent.

For a non-polar sorbent, it is preferable that a substrate issubstantially polar when the product produced by the enzyme issubstantially non-polar.

If the substrate is sufficiently polar, then the substrate present in apolar environment will not be retained on the non-polar sorbent and willbe removed from the sorbent column by the flow stream. Substratemolecules that have some non-polar regions may require the use ofsolutions that are made more non-polar so that they are not retained onthe sorbent bed. A solution can be made more non-polar, for example, bythe addition of methanol, acetonitrile, or tetrahydrofuran to water. Anon-polar product can be retained on a non-polar sorbent, such as C18,without the unreacted substrate being retained. The product is theneluted off the sorbent- using a non-polar elution solvent, such asmethanol.

More specifically, in one embodiment of the invention, the first columncontaining either antibodies or nucleic acid strands bound to a solidsupport is placed into an open ended column. The first column is placedinto a fluidics system such that sample and reagents are permitted toflow into the column to contact the solid support surface. If a sandwichtype immunoreaction or hybridization reaction is performed, for example,the sample is passed over the support and the target antibody or targetnucleic acid strand is captured. The enzyme reporter molecule is reactedwith specific material on the solid phase to determine if any materialhas been captured. This procedure requires the following steps: (a) washthe solid support to remove unreacted materials; (b) add the enzymelabeled antibody or polynucleotide probe; (c) wash the solid support toremove all unbound enzyme-labeled reagent; (d) add substrate specificfor the enzyme label; and (e) incubate the substrate and enzyme underappropriate conditions. If enzyme remains bound onto the solid phase,then the enzyme converts some portion of the substrate to product. Theamount of product produced is proportional to the amount of enzymeresiding on the solid phase.

In the fluidics system, the second column is located at a fixed distancefrom the first column. If the fluidics system is part of amicroprocessor-controlled instrument, then a switching valve may belocated between the two columns. This is the two column design as shownin FIG. 2. Alternatively, the columns may be constructed as a singleunit but with the solid support materials and the sorbent bed connectedat a fixed distance with a conduit or separated by a barrier, such as afrit or a membrane filter. This is a continuous design as is shown inFIG. 1.

When using either the two column design or the continuous design, thesubstrate is added to the solid support of the first column and allowedto incubate as described above. Preferably, the temperature of thesubstrate within the first column is regulated by a heating jacket orblock around the first column to obtain optimal enzyme activity. Afteran incubation time that can vary from two minutes to about 2 or morehours, the enzyme generated product is moved from the solid phase of thefirst column to the sorbent bed of the second column by a fluidicssystem.

The manner in which substrate is added to the solid phase in the finestcolumn and allowed to incubate with enzyme can be varied to optimizeenzyme activity. The method by which substrate is added and incubationoccurs, in turn, dictates the manner that product is brought to thesorbent bed in the second column. Four representative examples include:

1. The dynamic addition and incubation of substrate. In this embodiment,the substrate is added to the first column with continuous flow. Theflow out of the first column, containing substrate and product, isapplied by a continuous flow to the sorbent bed of the second column.After a defined period of continuous flow, wherein a defined amount ofsubstrate has been added to the first column, substrate addition isterminated. The columns may be washed with a non-eluting solution toremove substantially all of the substrate from the two column system.The product is removed from the second column by an elution solvent. Theconcentrated product is then measured by an appropriate detector, suchas a spectrophotometer, fluorometer, luminometer, or electrochemicaldetector. The advantages of dynamic addition and incubation are thatdiffusional constraints associated with enzyme activity on the solidphase are minimized and the accumulation of product in the first columnthat may cause feedback inhibition of the enzyme is eliminated.

2. Single stopped flow cycle. In this embodiment, substrate is added tothe solid phase of the first column and the flow is stopped. Incubationof the substrate with the enzyme occurs statically. After theappropriate incubation time, the substrate and product in the solventare moved to the sorbent bed of the second column by the fluidic system.The product remains bound to the sorbent bed while the substrate isremoved. The product can be removed from the sorbent bed by anappropriate elution solvent.

3. Multiple stopped flow cycles. In this embodiment, multiple additionsof substrate are made to the solid phase in the first column. Afterstatic incubation for a fixed period of time, the solid support columnis replenished by the addition of new substrate in solvent. The"incubated" substrate is moved into or toward the sorbent bed in thesecond column by the fluidics system. Since the fluidics system is aclosed system, the incubated substrate and product are moved through thesorbent bed in the second column with each sequential addition of thesubstrate solution to the first column. The product is retained on thesorbent bed while the substrate is substantially not retained. Whensubstrate addition is completed, the remaining substrate and product ismoved into and through the sorbent column. The product can then beeluted from the sorbent bed by the addition of an elution solvent intothe second column. The multiple stopped flow embodiment minimizes theproblems associated with product feedback inhibition of enzyme activity.Further, the product is concentrated into a small volume relative to thesubstrate volume.

4. Recycling of substrate. In this embodiment, substrate is added to thesolid support in the first column and continuously recirculated throughthe first column to provide dynamic incubation of the substrate with theenzyme on the solid phase. As a corollary, the recirculation can alsooccur from the effluent of the second column. In this approach,substrate is added to the first column, connected in series to thesecond column. The flow of solvent containing substrate and productproceeds continuously through the first column and into the secondcolumn wherein the product is captured in the sorbent bed. The substratepasses through the second column and is recirculated back into the firstcolumn for reaction with enzyme. The recirculation systems may bebeneficial when the substrate is expensive. Further, the continuouscirculation minimizes diffusion constraints associated with enzymeactivity on a solid support surface. After an appropriate period ofrecirculation, the product is eluted from the second column with anappropriate elution solvent.

Irrespective of the method of substrate addition and incubationdescribed, all embodiments result in the separation of enzyme-generatedproduct from substrate and concentration of the product for moresensitive detection.

The following examples illustrate the inventive process in an inventivedevice when used for a nucleic acid hybridization assay in Example 1 andfor an immunoassay in Example 2. Example 3 illustrates the use of theinvention for the detection of microbial contamination and Example 4illustrates the use of the invention for the detection of free enzyme ina sample.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1

This example describes a sandwich hybridization assay using anenzyme-labeled probe that is complementary to a sequence totallycontained within the LT gene of entertoxigenic strains of Escherichiacoli. An enzyme labeled DNA robe is made with B-galactosidase as theenzyme label on a single-stranded 26-met oligonucleotide probe. Theβ-galactosidase is bound to the probe by the procedure of Jablonski etal., Nucleic Acids Research 14:6115-28, 1986. A 26-met complement to theenzyme-labeled probe is linked via the 5' end to carboxy-modified latexbeads (0.97 μM from Interfacial Dynamics) through a 5-mer polyadenylatelinker arm by the method of Ghosh et al., Nucleic Acids Research15:5353-72, 1987. 50 μl of the latex bead suspension is hybridized withvarying concentrations of the enzyme-labeled probe. Hybridizationconditions are 37° C. for 1 hour in 0.5 ml of 5×SSPE (4.35% NaCl, 0.69%Na₂ H₂ PO₄, 0.185% EDTA, pH 7.4) and 0.1% SDS (sodium dodecyl sulfate).The latex beads are twice washed by sedimenting the beads with gentlecentrifugation for 10 minutes, followed by washing with 2×SSC (1.75%NaOH, 0.88% sodium citrate, pH 7.0) and 0.1% SDS.

The latex beads are added to the first column if the hybridization wasnot already conducted in the first column. Substrate,methylumbelliferyl-β-D-galactoside (MUGAL) at a concentration of 0.5μg/ml in 0.1 M phosphate buffer (pH 7) is added to the first column andincubated statically at 37° C. for 20 minutes. Incubation of MUGAL withthe bound marker enzyme, β-galactosidase, generates methylumbelliferone(MU) as the product. After incubation the substrate and product (MUGALand MU) are moved in 30% methanol in water by the fluidics system to thesecond column. The rate of flow of the fluidics system is 0.5 ml/min.

The second column contains the C18 non-polar sorbent PRP-1 (Hamilton)and packed into a column 0.074 inches (i.d.)×0.75 inches (L) withapproximately 50 to 200 theoretical plates. The MU product will beretained in the sorbent bed, while the more polar MUGAL will not beretained on the sorbent when water is the solvent. Elution andconcentration of the MU product from the second column is accomplishedwith 100% methanol. The concentration of MU is detected by a flowfluorometer (Kratos, Spectro flow 980). The amount of MU produced isdirectly proportional to the amount of target DNA in the sample that hasbeen captured onto the solid support.

EXAMPLE 2

This example describes a heterogeneous competitive enzyme immunoreactionwith thyroxine (T4) using fluorescent detection. Monoclonal antibodies(Immunosearch, Toms River, N.J.) to T4 are covalently linked viacarbodiimide coupling to a carboxy-modified latex beads using the methodof Quash et al. J. Immunological Methods 22:165-74 (1978). The solidsupport (latex beads) is added to the first column. A standardizedamount of alkaline phosphatase labeled T4 antigen, which is reactivewith the antibody on the solid support, is mixed with a samplecontaining the unlabeled T4 antigen in a 0.1M phosphate buffer (pH 7).The sample containing the labeled and unlabeled T4 antigens isintroduced into the first column containing the antibody coated latexbeads. The labeled and unlabeled T4 antigens compete for antibodybinding sites. The amount of labeled antigen remaining on the solidsupport surface is inversely proportional to the amount of unlabeledantigen in the sample. Unreacted materials are removed from the firstcolumn with a wash using a 0.1M phosphate buffer (pH 7). The substrate,0.5 μg/ml 4-methyl-umbelliferyl phosphate (MUP) in 0.1M Tris buffer with0.1M NaCl and 50 mM magnesium chloride (pH 8.5) is added to the firstcolumn and allowed to incubate for 5-20 min at 37° C. incubation of thesubstrate with the enzyme on the solid support generatesmethylumbelliferone (MU) as a product. After incubation, the substrateand product solution is moved by a fluidics system to a second column.The second column contains the C18 non-polar sorbent PRP-1, which ispacked in a column 0.074 inch (i d )×0.75 inch (length). This produces acolumn with approximately 50 theoretical plates. The product is retainedon the C18 sorbent, while the substrate is not retained due todifferences in hydrophobicity. MU is removed from the second column withan elution, solvent consisting of is 100% methanol and measured in aflow fluorometer (Kratos, Spectroflow 980).

EXAMPLE 3 Microbial Detection and Estimation Tests in Samples

Many different types of samples such as food, water, wastewater, dairy,clinical and pharmaceutical samples may be tested for microbialcontaminants essentially using the enzyme detection procedure describedin the present invention. The microbial contaminants could includespecific microorganisms such as Escherichia coli or groups ofmicroorganisms such as the coliform bacteria, the fecal coliformbacteria, the total count or heterotrophic bacteria, yeasts, and molds.Detection and estimation of these microbial contaminants would beaccomplished by assaying for an enzyme or enzymes produced by thesemicroorganisms. The use of sorbents in an inventive device providesearlier testing results by (i) separating the product of the enzymereaction from the substrate and soluble sample constituents using asorbent bed in a column and (ii) concentrating the product from theassay solution into a small detection volume.

Coliform bacteria produce the enzyme β-galactosidase for utilization oflactose. The detection and measurement of activity associated with thisenzyme can be used to rapidly detect and estimate the levels of thesebacteria in a sample such as food. This is accomplished by adding a foodsample (25 grams) into a broth culture medium (225 ml) supplemented withthe fluorogenic substrate 4-methylumbelliferyl-β-D-galactoside (MU-GAL)at a level of approximately 50-100 μg/ml. The preferential broth culturemedium is one which permits the growth of the coliform bacteria whileinhibiting or suppressing the growth of non-coliform bacteria. Examplesare commonly used media are violet red bile broth, Endo broth, or laurylsulfate broth or less commonly used formulations such as CM (withoutagar) (Firstenberg-Eden, R. and Klein, C. S., J. Food Science 48:1307,1983). The fluorogenic substrate MU-GAL is cleaved by the cellularβ-galactosidase enzyme to produce methylumbelliferone and galactoside.After a specified incubation period, preferably 1-6 hours, at 35° C., asmall aliquot of the culture medium is transferred to a columncontaining reverse phase C18 sorbent. The methylumbelliferone stronglybinds to the reverse phase sorbent, whereas sample constituents andremaining substrate, under the appropriate solution conditions, do notsubstantially bind. The methylumbelliferone remaining on the sorbent iseluted from the sorbent with the appropriate elution solvent andmeasured using a fluorometer. The amount of fluorescence is proportionalto the number of coliform bacteria in the sample after incubation. Thelevel of coliform bacteria initially in the sample is estimated from astandard curve that plots (i) relative fluorescence units versus initialconcentration of bacteria for a specified incubation time or (ii) time(hours) to detect fluorescence versus initial concentration of bacteria.

EXAMPLE 4 Detection of Free Enzyme in a Sample

The detection and measurement of free enzymes in a sample are used for avariety of purposes ranging from tests for food and dairy safety andproduct quality to tests for clinical diagnosis. Examples of assays forfree enzymes in food and dairy products include the determination of (i)pasteurization completeness by measuring activity of alkalinephosphatase in fluid milk and other dairy products and (ii) spoilagepotential (product shelf-life) by measuring the activity of enzymes suchas protease enzymes, trimethylamine oxidase, xanthine oxidase orcytochrome enzymes such as cytochrome b5 reductase. Clinical diagnostictests include assays for creatine kinase activity to assess for damageto the myocardium, alkaline phosphatase activity in serum forhepatobiliary diseases and bone diseases, and lactate dehydrogenaseactivity in cerebrospinal fluid and serum to determine tissue damage.

Adequate pasteurization results in the inactivation of the enzymealkaline phosphatase. Therefore an alkaline phosphatase test in fluidmilk measures pasteurization efficacy. A milk sample is added to acarbonate-magnesium buffered solution supplemented with the fluorogenicsubstrate 4-methylumbelliferyl phosphate (MUP); The alkaline phosphatasecleaves the MUP to produce methylumbelliferone and phosphate. Thissolution is incubated under appropriate conditions and an aliquot istransferred to a column of the present invention, containing a C18reverse phase sorbent. The methylumbelliferone is retained on the C18sorbent while the sample constituents and remaining substrate are notretained under appropriate solution conditions. The methylumbelliferoneis eluted from the sorbent with the appropriate solvent and thefluorescence measured. The amount of fluorescence is proportional to theamount of alkaline phosphatase in the sample.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

We claim:
 1. A method for determining the presence or concentration ofan enzyme marker bound to an antibody, antigen or strand of nucleic acidgenerated in a hybridization assay or immunoassay, comprising the stepsof:adding a substrate in a selected solution to a first columncontaining the enzyme marker further bound to a solid phase as a resultof the hybridization assay and immunoassay, said substrate beingreactive with said bound enzyme marker; incubating the first column toenzymatically convert said substrate to a product in an amountproportional to the amount of bound enzyme marker present; transferringthe product and unreacted substrate onto a second column of no more than500 theoretical plates, said second column containing sorbentselectively binding said product in the presence of said selectedsolution; eluting the product from the sorbent; and detecting thepresence or concentration of the product.
 2. The method of claim 1wherein the solution containing the substrate flows through the firstcolumn and out the second column in a continuous manner.
 3. The methodof claim 1 wherein the solution containing the substrate flows throughthe first column and out the second column via a stopped flow cycle. 4.The method of claim 1 wherein the product is detected by absorbance,fluorescense, luminescence or by electrochemical means.